Gualberto Ruano

The Accuracy of Statistical Methods for Estimation of Haplotype Frequencies: An Example from the CD4 Locus

Haplotype analysis has become increasingly important for the study of human disease as well as for reconstruction of human population histories. Computer programs have been developed to estimate haplotype frequencies statistically from marker phenotypes in unrelated individuals. However, there currently are few empirical reports on the accuracy of statistical estimates that must infer linkage phase. We have analyzed haplotypes at the CD4 locus on chromosome 12 that consist of a short tandem-repeat polymorphism and an Alu insertion/deletion polymorphism located 9.8 kb apart, in 398 individuals from 10 geographically diverse sub-Saharan African populations. Haplotype frequency estimates obtained using gene counting based on molecularly haplotyped (phase-known) data were compared with haplotype frequency estimates obtained using the expectation-maximization algorithm. We show that the estimated frequencies of common haplotypes do not differ significantly with the use of phase-known versus phase-unknown data. However, rare haplotypes are occasionally miscalled when their presence/absence must be inferred. Thus, for those research questions for which the common haplotypes are most important, frequency estimates based on the phase-unknown marker-typing results from unrelated individuals will be sufficient. However, in cases where knowledge of rare haplotypes is critical, molecular haplotyping will be necessary to determine linkage phase unambiguously.

Coupled amplification and sequencing of genomic DNA.

Addition of dideoxyribonucleotides during the exponential phase of the PCR should result in the synthesis of two complementary sequence ladders. We have explored this hypothesis to develop coupled amplification and sequencing of genomic DNA. Coupled amplification and sequencing is a biphasic method for sequencing both strands of template as they are amplified. Stage I selects and amplifies a single target from the genomic DNA sample. Stage II accomplishes the sequencing as well as additional amplification of the target using aliquots from the stage I reaction mixed with end-labeled primer and dideoxynucleotides. We have successfully applied coupled amplification and sequencing to a 300-base-pair fragment 4 kilobases upstream from HOX2B directly from human whole genomic DNA.

Distribution and frequency of a polymorphic Alu insertion at the plasminogen activator locus in humans

We have investigated the frequency distribution, across a broad range of geographically dispersed populations, of alleles of the polymorphicAlu insertion that occurs within the 8th intron of the tissue plasminogen activator gene (PLAT). ThisAlu is a member of a recently derived subfamily ofAlu elements that has been expanding during human evolution and continues to be transpositionally active. We used a “population tube” approach to screen 10 chromosomes from each of 19 human populations for presence or absence of thisAlu in the PLAT locus and found that all tested populations are dimorphic for presence/absence of this insertion. We show that the previously publishedEcoRI,HincII,PstI,TaqI, andXmnI polymorphisms at the PLAT locus all result from insertion of thisAlu and we use both restriction fragment length polymorphism and polymerase chain reaction analysis to examine the frequency ofAlu(+) andAlu(−) alleles in a sample of 1003 individuals from 27 human populations and in 38 nonhuman primates. Nonhuman primates are monomorphic for theAlu(−) allele. Human populations differ substantially in allele frequency, and in several populations both alleles are common. Our results date the insertion event prior to the spread and diversification of modern humans.

Studies on DNA polymorphisms in human bone and soft tissues

Abstract The determination of genetic characteristics in human bone and soft tissues is interest both to forensic scientists and to physical anthropologists, although for different purposes. The application of genetic typing from human remains to anthropological and forensic identification problems is briefly reviewed. DNA analyses of ancient and modern human remains by Southern blot (RFLP) and amplification (PCR) techniques are reviewed. The results of studies on the effects of exposing bone and soft tissues to different environmental conditions for defined time periods on subsequent DNA typing are presented. Bone specimens were temperatures and soil under dry and humid conditions, and a complete series of soft tissues were exposed to dry and moist air and to salt water, for periods of 1–9 months.

DNA analysis in human bone and other specimens of forensic interest: PCR typing and testing

Polymerase chain reaction (PCR) procedures that enable the faithful replication of millions of copies of a specific DNA sequence in vitro have been described and refined in recent years [I]. PCR techniques have been used in a variety of genetic and clinical diagnostic applications, including evolutionary studies, cloning and sequencing, detection of sickle cell anaemia genes, detection of human papilloma virus, HIV and other retroviruses. Because many specimens submitted for forensic DNA analysis contain limited quantities of DNA and the DNA in these specimen may be degraded, RFLP analysis is not always possible. This limitation has prompted the interest of forensic scientists in the applicability of PCR typing procedures that may be applicable to small and/or degraded specimens. PCR techniques may be applied to forensic identification problems using specific primers for the amplification of coding loci, variable number of tandem repeats (VNTR) loci, or sequences specific for X and/or Y chromosomes that can serve as sex markers. The most refined PCR procedure applicable to forensic identification thus far utilized specific primers and allele-specific oligonucleotide probes to detect genotypes at the HLA-DQ, locus [2-41. HLA-DQ, typing techniques have been applied to blood and seminal stains [5] and to hair roots [6].

Detailed analysis of a 17q21 microdissection library by sequence bioinformatics and isolation of region-specific clones

A region-specific microdissection library originating from human chromosome 17q21, was constructed using the MboI linker-adaptor microcloning techique. DNA sequencing of 241 microclones resulted in the identification of 74 novel coding sequences, paralogs of known genes, and known, but previously unmapped, genes or expressed sequence tags that were “virtually” mapped to chromosome 17q21. By pooling the microclones as multiplexed hybridizaion probes, and by virtue of their origin on 17q21, we were able to identify approximately 150 P1 clones from the human Reference Library Data Base P1 Library that potentially map to chromosome 17q21. Verification of the 17q21 location of 16 P1 clones was accomplished by PCR analysis, with STS primer pairs to known 17q21 genes or by FISH. Our results demonstrate the substantial advantage of combining the sequence analysis of microclones with multiplex hybridization strategies for gene discovery and mapping specific gene rich regions of the genome.